Keeping blood in a fluid state, termed hemostasis, requires a subtle balance of pro- and anticoagulants. Procoagulants prevent excessive bleeding by blocking blood flow from a damaged vessel, whereas anticoagulants prevent clots from forming in the circulating system which could otherwise block blood vessels and lead to myocardial infarction or stroke.
The biochemical sequence leading to a blood clot is termed the coagulation cascade. The mechanism is based on catalytic conversion of fibrinogen, a soluble plasma protein, to insoluble fibrin. The enzyme catalyzing this reaction is thrombin, which does not permanently circulate in the blood in an active form but exists as prothrombin, the inactive precursor of thrombin. Conversion to thrombin occurs in the presence of calcium ions and tissue thromboplastin. This mechanism is known as the extrinsic pathway. A second, more complex, intrinsic pathway is activated by clotting factors associated with platelets and is well understood in the art.
Breakdown of a blood clot, termed fibrinolysis, requires the conversion of fibrin to a soluble product. This lysis is catalyzed by the proteolytic enzyme plasmin, which circulates in an inactive form, plasminogen. Tissue plasminogen activator (tPA), bacterial hemolytic enzymes (e.g., streptokinase), and proteolytic human enzymes found in urine (e.g., urokinase) all activate plasminogen. These materials are used in thrombolytic therapy.
Diagnosis of hemorrhagic conditions such as hemophilia, where one or more of the twelve blood clotting factors may be defective, can be achieved by a wide variety of coagulation tests. In addition, several tests have been developed to monitor the progress of thrombolytic therapy. Other tests have been developed to signal a prethrombolytic or hypercoagulable state, or monitor the effect of administering protamine to patients during cardiopulmonary bypass surgery. However, the main value of coagulation tests is in monitoring oral and intravenous anticoagulation therapy. Two of the key diagnostic tests are activated partial thromboplastin time (APTT) and prothrombin time (PT).
An APTT test evaluates the intrinsic and common pathways of coagulation. For this reason APTT is often used to monitor intravenous heparin anticoagulation therapy. Specifically, it measures the time for a fibrin clot to form after the activating agent, calcium, and a phospholipid have been added to the citrated blood sample. Heparin administration has the effect of suppressing clot formation.
A PT test evaluates the extrinsic and common pathways of coagulation and, therefore, is used to monitor oral anticoagulation therapy. The oral anticoagulant coumadin suppresses the formation of prothrombin. Consequently, the test is based on the addition of calcium and tissue thromboplastin to the blood sample.
The standard laboratory technology for coagulation tests typically uses a turbidimetric method (See, for example, U.S. Pat. No. 4,497,774). For analysis, whole-blood samples are collected into a citrate vacutainer and then centrifuged. The assay is performed with plasma to which a sufficient excess of calcium has been added to neutralize the effect of citrate. For a PT test, tissue thromboplastin is provided as a dry reagent that is reconstituted before use. This reagent is thermally sensitive and is maintained at 4 degrees C by the instruments. Aliquots of sample and reagent are transferred to a cuvette heated at 37 degrees C, and the measurement is made based on a change in optical density.
As an alternative to the turbidimetric method, Beker et al. (See, Haemostasis (1982) 12:73) introduced a chromogenic PT reagent (Thromboquant PT). The assay is based on the hydrolysis of p-nitroaniline from a modified peptide, Tos-Gly-Pro-Arg-pNA, by thrombin and is monitored spectrophotometrically.
Coagulation monitors are known for the analysis of whole blood. For example, a unit-use cartridge has been described in which dry reagents are placed into the analyzer which is then heated to 37 degrees C before a drop of blood is introduced. The sample is mixed with the reagent by capillary draw. The detection mechanism is based on laser light passing through the sample. Blood cells moving along the flow path yield a speckled pattern specific to unclotted blood. When the blood clots, movement ceases producing a pattern specific to clotted blood. Several patents disclose aspects of this technology and are described further, below.
U.S. Pat. No. 4,731,330 discloses a lyophilized whole-blood control sample consisting of fixed red blood cells and plasma solids including coagulation factors. Standard plasma controls cannot be used with this system because detection is based on the motion of red blood cells. U.S. Pat. No. 4,756,884 discloses the component parts of the cartridge technology, which is based on capillary draw, including certain antibodies and reagents for blood clotting. U.S. Pat. No. 4,948,961 discloses the components and method of use of an optical simulator cartridge used with the above instrument. U.S. Pat. No. 4,952,373 discloses a plastic safety shield that prevents blood from being inadvertently transferred from the blood entry port of the cartridge into testing region of the instrument.
U.S. Pat. No. 4,963,498 discloses a method of obtaining chemical information from the capillary draw cartridge. U.S. Pat. No. 5,004,923 discloses optical features by which the above instrument interrogates the cartridge. U.S. Pat. No. 5,039,617 discloses a device for APTT using whole-blood in which the sample is mixed with reagents as it is drawn by capillary action along a fluid path. The activating reagent, sulfatide or sulfoglycosylsphingolipid, provides a clot formation that is independent of hematocrit. EP 0368624 A2 discloses a method for providing dry, yet easily resuspendible, stable latex particles on a surface. EP 0395384 A2 discloses a coding method by which different types of cartridges can be identified.
A unit-use cartridge has also been described which comprises two capillary tubes that simultaneously draw blood from a single finger-stick. The design allows for duplicate measurement or two different measurements based on different reagent coatings. The PCT application WO 89/06803 describes the above apparatus for measuring blood coagulation based on changes in light permeability through a capillary tube.
On the other hand, U.S. Pat. No. 3,695,842 describes a method of analyzing the transformation of a liquid to a gelatinous or solid mass and is applied to PT and APTT. The coagulation system uses a vacutainer that contains all the necessary reagents, as well as a ferromagnetic component. Once the blood sample has been drawn into the vacutainer, it is placed into the instrument in an inclined manner. This procedure makes the ferromagnetic component sit at the bottom of the tube in close proximity to a magnetic reed switch. As the sample is rotated, gravity ensures that the component remains close to the reed switch. However, as the blood starts to clot, viscosity increases to the point where the component starts to rotate with the blood sample. The reed switch is thus activated, enabling a coagulation time to be estimated.
Yet another format has been described based on the use of magnetic particles mixed into a dry reagent contained within a flat capillary chamber. An applied oscillating magnetic field from the instrument causes the particles to oscillate once the reagent has dissolved in the blood. This motion is monitored optically. When the blood clots, the particles become entrapped and motion is diminished. Fibrinolysis assays are performed by monitoring the reverse process (See, Oberhardt et al., Clin, Chem. (1992) 37:520). The above magnetic particle-based method is also described in U.S. Pat. No. 5,110,727.
Another method of detecting coagulation is based on ultrasound scattering from 200 micron glass spheres suspended in a blood sample. Amplitude and phase changes of the scattered waves are used to detect coagulation (See, Machado et al., J. Acoust. Soc. Am. (1991) 90:1749).
Shenaq and Saleem, in "Effective Hemostasis in Cardiac Surgery," Eds: Ellison, N. and Jobes, D. R., Saunder & Co., (1988), utilize a sonic probe that is inserted into a cuvette containing the sample and reagents. The sonic probe responds to clot formation in the cuvette and, thus, can be used to measure the coagulation time.
An automatic coagulation timer has been described which measures the activated clotting time (ACT) in blood samples from patients during cardiopulmonary bypass. The sample is added to a cartridge which incorporates a stirring device on to which the clot forms. Motion of the stirring device is controlled by a photo optical detector (See, Keeth et al., Proceedings Am. Acad. Cardiovascular Perfusion (1988) 9:22).
An instrument for automatically recording clot lysis time based on a change in electrical conductivity has been reported (See, Wilkens and Back, Am. J. Clin. Pathol. (1976) 66:124). In the first step, streptokinase, thrombin and fibrinogen are added to a tube. Once a clot is formed the tube is partially inverted so that as streptokinase dissolves the clot, solution drains down the tube and drips into a second tube that has one electrode positioned at the bottom and another further up the tube. When the fluid reaches the second electrode a digital timer is stopped yielding an estimate of the clot dissolution time.
Another method of measuring clot lysis time based on breaking an electrical circuit maintained by a single fibrin strand has been reported (See, Folus and Kramer, J. Clin. Pathol. (1976) 54:361).
The disclosure of U.S. Pat. No. 5,096,669 includes the general format for blood chemistry testing (e.g., potassium and glucose blood levels) and the use of a bladder to move a sample fluid to a sensor region in a single direction. In particular, variation of the speed or direction of motion or the oscillation of a fluid sample is nowhere disclosed, taught or suggested.
Therefore, there remains a need for an apparatus and method of conducting assays that are responsive to changes in the viscosity of a fluid sample, which apparatus and method can be used at the point of care, especially locations, such as a doctor's office, which have no immediate access to a centralized testing facility, and which apparatus and method can be optionally produced by microfabrication methods and be readily adapted to include a multiplicity of tests, including blood gas and analyte testing.